Comprehensive Essentiality Analysis of the Mycobacterium tuberculosis Genome via Saturating Transposon Mutagenesis
For decades, identifying the regions of a bacterial chromosome that are necessary for viability has relied on mapping integration sites in libraries of random transposon mutants to find loci that are unable to sustain insertion. To date, these studies have analyzed subsaturated libraries, necessitat...
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2017-01-01
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doaj-b5d9e3185d1d4f53a056360ca88e56cc2021-07-02T09:01:31ZengAmerican Society for MicrobiologymBio2150-75112017-01-0181e02133-1610.1128/mBio.02133-16Comprehensive Essentiality Analysis of the Mycobacterium tuberculosis Genome via Saturating Transposon MutagenesisMichael A. DeJesusElias R. GerrickWeizhen XuSae Woong ParkJarukit E. LongCara C. BoutteEric J. RubinDirk SchnappingerSabine EhrtSarah M. FortuneChristopher M. SassettiThomas R. IoergerChristina L. StallingsFor decades, identifying the regions of a bacterial chromosome that are necessary for viability has relied on mapping integration sites in libraries of random transposon mutants to find loci that are unable to sustain insertion. To date, these studies have analyzed subsaturated libraries, necessitating the application of statistical methods to estimate the likelihood that a gap in transposon coverage is the result of biological selection and not the stochasticity of insertion. As a result, the essentiality of many genomic features, particularly small ones, could not be reliably assessed. We sought to overcome this limitation by creating a completely saturated transposon library in Mycobacterium tuberculosis. In assessing the composition of this highly saturated library by deep sequencing, we discovered that a previously unknown sequence bias of the Himar1 element rendered approximately 9% of potential TA dinucleotide insertion sites less permissible for insertion. We used a hidden Markov model of essentiality that accounted for this unanticipated bias, allowing us to confidently evaluate the essentiality of features that contained as few as 2 TA sites, including open reading frames (ORF), experimentally identified noncoding RNAs, methylation sites, and promoters. In addition, several essential regions that did not correspond to known features were identified, suggesting uncharacterized functions that are necessary for growth. This work provides an authoritative catalog of essential regions of the M. tuberculosis genome and a statistical framework for applying saturating mutagenesis to other bacteria.http://mbio.asm.org/cgi/content/full/8/1/e02133-16 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Michael A. DeJesus Elias R. Gerrick Weizhen Xu Sae Woong Park Jarukit E. Long Cara C. Boutte Eric J. Rubin Dirk Schnappinger Sabine Ehrt Sarah M. Fortune Christopher M. Sassetti Thomas R. Ioerger Christina L. Stallings |
spellingShingle |
Michael A. DeJesus Elias R. Gerrick Weizhen Xu Sae Woong Park Jarukit E. Long Cara C. Boutte Eric J. Rubin Dirk Schnappinger Sabine Ehrt Sarah M. Fortune Christopher M. Sassetti Thomas R. Ioerger Christina L. Stallings Comprehensive Essentiality Analysis of the Mycobacterium tuberculosis Genome via Saturating Transposon Mutagenesis mBio |
author_facet |
Michael A. DeJesus Elias R. Gerrick Weizhen Xu Sae Woong Park Jarukit E. Long Cara C. Boutte Eric J. Rubin Dirk Schnappinger Sabine Ehrt Sarah M. Fortune Christopher M. Sassetti Thomas R. Ioerger Christina L. Stallings |
author_sort |
Michael A. DeJesus |
title |
Comprehensive Essentiality Analysis of the Mycobacterium tuberculosis Genome via Saturating Transposon Mutagenesis |
title_short |
Comprehensive Essentiality Analysis of the Mycobacterium tuberculosis Genome via Saturating Transposon Mutagenesis |
title_full |
Comprehensive Essentiality Analysis of the Mycobacterium tuberculosis Genome via Saturating Transposon Mutagenesis |
title_fullStr |
Comprehensive Essentiality Analysis of the Mycobacterium tuberculosis Genome via Saturating Transposon Mutagenesis |
title_full_unstemmed |
Comprehensive Essentiality Analysis of the Mycobacterium tuberculosis Genome via Saturating Transposon Mutagenesis |
title_sort |
comprehensive essentiality analysis of the mycobacterium tuberculosis genome via saturating transposon mutagenesis |
publisher |
American Society for Microbiology |
series |
mBio |
issn |
2150-7511 |
publishDate |
2017-01-01 |
description |
For decades, identifying the regions of a bacterial chromosome that are necessary for viability has relied on mapping integration sites in libraries of random transposon mutants to find loci that are unable to sustain insertion. To date, these studies have analyzed subsaturated libraries, necessitating the application of statistical methods to estimate the likelihood that a gap in transposon coverage is the result of biological selection and not the stochasticity of insertion. As a result, the essentiality of many genomic features, particularly small ones, could not be reliably assessed. We sought to overcome this limitation by creating a completely saturated transposon library in Mycobacterium tuberculosis. In assessing the composition of this highly saturated library by deep sequencing, we discovered that a previously unknown sequence bias of the Himar1 element rendered approximately 9% of potential TA dinucleotide insertion sites less permissible for insertion. We used a hidden Markov model of essentiality that accounted for this unanticipated bias, allowing us to confidently evaluate the essentiality of features that contained as few as 2 TA sites, including open reading frames (ORF), experimentally identified noncoding RNAs, methylation sites, and promoters. In addition, several essential regions that did not correspond to known features were identified, suggesting uncharacterized functions that are necessary for growth. This work provides an authoritative catalog of essential regions of the M. tuberculosis genome and a statistical framework for applying saturating mutagenesis to other bacteria. |
url |
http://mbio.asm.org/cgi/content/full/8/1/e02133-16 |
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